The measurement of the pH of biological fluids is an important aspect of a variety of assays. In particular, intracellular pH plays an important modulating role in many cellular events, including cell growth, calcium regulation, enzymatic activity, receptor-mediated signal transduction, ion transport, endocytosis, chemotaxis, cell adhesion, and other cellular processes. The use of fluorescence-based techniques may increase the sensitivity of measurements and allow the measurement of intracellular pH in single cells, for example, by flow cytometry or existing fluorescence-based automated high-throughput microplate assays. Imaging techniques that use fluorescent pH indicators also may allow researchers to investigate these processes with much greater spatial resolution and sampling density than can be achieved using other available technologies, such as microelectrodes. Fluorescence assays are typically able to utilize low concentrations of the pH indicator, potentially reducing toxicity and buffering effects.
In selecting a pH indicator, to allow the greatest sensitivity to small changes in pH of the medium, the equilibrium constant between the acidic and basic forms of the indicator for the dye (i.e., the pKa) should be near the pH of the selected assay medium. For physiological assays, such as blood and most intracellular fluid assays, the pH typically is in the range pH 6 to pH 8, and more typically in the range pH 6.5 to 7.6.
A variety of fluorescein-based pH indicators have been described, including fluorescein, carboxyfluorescein, fluorescein sulfonic acid, chloromethylfluorescein, carboxynaphthofluorescein, seminaphthorhodafluor derivatives, and seminaphthofluorescein derivatives. Typically, for intracellular assays, the dye is used as a cell-permeant diacetate derivative that is subsequently cleaved by nonspecific esterases within the cells of the sample, producing the active indicator.
Perhaps the most commonly utilized fluorescein-based pH indicator is 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, more commonly known as BCECF. BCECF has a pKa of 7.0, making it an ideal choice for intracellular assays. In addition, BCECF exhibits pH-dependent dual excitation and is excited efficiently by the 488-nm line of an argon-ion laser, as used in a variety of instruments. Ester derivatives of BCECF, most typically BCECF-AM, are nonfluorescent and membrane-permeant, and can be loaded into cells without disruption of the cell membranes. The conversion to the fluorescent version of the indicator can serve as an indicator of cell viability, as well as permitting subsequent pH measurements.
Unfortunately, for all its advantages, BCECF also possesses significant disadvantages. The synthesis of BCECF-AM typically produces a mixture of discrete compounds, as shown below in formulas I, II, and III.
This results in substantial variations in the ratios of active components and the profiles of impurities. Thus, different commercial sources of BCECF-AM commonly yield indicators of highly variable quality due to differences in their manufacturing processes. In fact, the quality often varies considerably from batch to batch even from a single manufacturer. The variation of component ratios has caused some difficulties in obtaining reproducible data due to the lack of consistent commercial material. In addition, the extremely weak excitation peak of BCECF at shorter wavelengths makes excitation-ratiometric measurements difficult, and it is impossible to perform emission-ratiometric pH measurements using BCECF due to the lack of pH-dependent dual emission. Thus, there is a need for improved fluorescent pH indicators, particularly for intracellular assays.